1. Field of the Invention
This invention relates to a process for the solid-phase synthesis of peptide amides, to Fmoc-α-nitrogen protected Cα-carboxamide amino acids, novel peptide amides and novel supports for peptide amide synthesis.
2. Description of Related Art
Many peptides have found utility as therapeutic agents. One peptide that is of particular importance is eptifibatide a cyclic peptide amide containing six amino acids and one mercaptopropionyl residue. Eptifibatide acts by binding to the platelet receptor glycoprotein IIb/IIIa and inhibiting platelet aggregation, thus preventing blood clots. Eptifibatide is the active agent in Integrelin™, which is used in treating patients with acute coronary syndrome and those undergoing percutaneous coronary intervention.
A solid-phase method for the production of eptifibatide is described in U.S. Pat. No. 5,318,899. However, as with many peptides the major challenge with eptifibatide is to produce sufficient material at an acceptable cost.
Peptides may be synthesised by solid-phase synthesis that commences from the carboxy-terminal end of the peptide using an alpha-amino protected amino acid. The two most widely used protocols employ tert-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) as amino protecting groups.
When an Fmoc protection strategy is employed the favoured solid-phase support is a polydimethylacrylamide or polystyrene resin derivatised with a 4-(2′,4′-dimethoxybenzyl-Fmoc-aminomethyl)-phenoxy (Fmoc-Rink amide) linkage agent. The first protected amino acid of the peptide sequence may be attached to this resin by converting its carboxyl group to an active ester, usually by reacting with N-hydroxybenzotriazole (BtOH) in the presence of an activating agent such as diisopropylcarbodiimide (DIC). This mild activation step minimises racemisation of susceptible residues such as Cys and Trp (throughout this specification standard three letter or single letter abbreviations for amino acids will be used). In addition, mild acidolysis of this peptide resin can result in both the cleavage of the peptide from the resin and the deprotection of any side chain protecting groups. However, the Fmoc-Rink amide linkage agent is an expensive reagent and this limits its use in commercial-scale peptide synthesis.
An alternative strategy is to use a resin bearing a 4-hydroxymethylbenzoic acid (HMBA) linkage unit. These derivatised resins are cheaper than those derivatised with the Fmoc-Rink amide linker. However, attachment of the first amino acid involves a p-dimethylaminopyridine (DMAP) catalysed esterification protocol that can result in both incidental Fmoc removal and racemisation of susceptible amino acids (Atherton, E. et al. (1981) J. Chem. Soc. Chem. Commun. p 336).
Peptides are usually converted to the corresponding amides by ammonolysis/aminolysis of resin bound peptide esters or by synthesis of the peptide amide using an amine based linker such as the Fmoc-Rink amide resin. Thus their production is also limited by the factors outlined above.
Therefore there is a requirement for a process by which peptide amides such as eptifibatide may be economically produced in high purity on a large scale.